This invention relates to the control of plant hormone levels and of plant growth at the molecular genetic level. It particularly relates to nucleotide sequences encoding UDP-glucose:indol-3-ylacetyl-glucosyl transferase, and the use of these sequences and/or subsequences thereof to regulate plant growth.
Plant growth is affected by a variety of physical and chemical factors. Physical factors include available light, day length, moisture and temperature. Chemical factors include minerals, nitrates, hormones and cofactors.
One of the most common plant growth hormones is indole-3-acetic acid (IAA). IAA is often referred to as xe2x80x9cauxin.xe2x80x9d IAA has been demonstrated to be directly responsible for increase in growth in plants in vivo and in vitro. Those characteristics influenced by IAA include cell elongation, internodal distance (height), leaf surface area and crop yield.
Most plant tissues contain about 10xe2x88x928 M free IAA. There appears to be two basic pathways for the synthesis of IAA in plants, one via tryptophan and one probably through indole. These same tissues contain about 20 times that amount of IAA in the form of ester or amide conjugates; most commonly the IAA is covalently bound to a sugar moiety. This 20:1 ratio of conjugated to free IAA is generally observed even in tissues which are known to be limited in growth rate by the amount of free IAA.
The first step in the biosynthesis of conjugates of IAA in Zea mays is catalyzed by UDP-glucose:indol-3-ylacetyl-glucosyl transferase (EC 2.4.1.121; also called IAA-Glucose Synthetase, IAGlu Synthetase, IAGlu Transferase). This enzyme has been purified, and its characteristics have been described (Kowalczyk and Bandurski (1991) Biochem. J. 279:509-514; Leznicki and Bandurski (1988) Plant Physiol. 88:1481-1485 and 88:1474-1480). The substrates for IAGlu Transferase are UDP-glucose and IAA, and the reaction product is 1-0-xcex2-D-indol-3ylacetyl-glucose. IAA-glucose can be hydrolyzed by one of two hydrolases, depending on the isomeric form. These hydrolases effectively impart reversibility to the synthetase reaction.
IAGlu is an acyl alkyl acetal, and its energetically unfavorable synthesis is followed by an energetically favorable transacylation of IAA from IAGlu to myo-inositol to yield indol-3-ylacetyl-myo-inositol (Michalczuk and Bandurski (1982) Biochem. J. 207: 273-281). The enzyme indol-3-ylacetylglucose-myo-inositol indol-3-ylacetyltransferase (IAInos synthetase) catalyzes this reaction (Reaction D, FIG. 1). IAInos is believed to be a transport form of IAA, and IAInos is the substrate for the synthesis of IAInos-glycosides. Thermodynamically, IAInos synthetase is believed to be the enzyme which shifts the equilibrium from free IAA to conjugated forms of IAA. Conjugates appear to serve functions other than growth promotion such as IAA transport (Nowacki and Bandurski (1980) Plant Physiol. 65:422), protection of IAA against peroxidative attack (Cohen and Bandurski (1978) Planta 139:203), storage of IAA in seeds (Bandurski et al. (1991) in Plant Growth Substances, C. M. Karssen (ed.), Kluwer Academic Publishing, Amsterdam, pp. 1-12) and hormonal homeostasis (Bandurski et al. (1988) in Plant Growth Substances, Pharis and Rood (eds.), Springer-Verlag, Berlin, pp. 341-352).
There have been attempts to improve crop yield by increasing the level of IAA in plants both by application of exogenous IAA and by increasing the synthesis of endogenous IAA. Yang et al. (1993) Plant Physiol. 102:717-724 report that exogenously applied IAA, via cotton wicking in contact with apical stem parts, stimulated stem elongation, particularly in dwarf plants. Application of exogenous IAA is not practical because the effect is limited in time and such application at the agricultural level would be prohibitively labor-intensive and expensive.
Attempts to increase the endogenous synthesis of IAA have involved the genetic engineering of plants to contain bacterial genes for the biosynthesis of IAA. There have been several reports that expression of the Agrobacterium tumefaciens IAA biosynthetic pathway genes did not result in increased plant growth (Follin et al. (1985) Mol. Gen. Genet. 201:178-185; van Onckelen et al. (1985) FEBS Letters 181:373-376). Generally transgenic plants expressing higher levels of IAA via bacterial enzymes showed phenotypic abnormalities (Klee et al. (1987) Genes Devel. 1:86-96; Schmulling et al. (1988) EMBO J. 7:2621-2629). Such transgenic plants exhibited higher than normal levels of both IAA conjugates and of free IAA, particularly when the bacterial iaaM and/or iaaH genes were linked to powerful heterologous promoters (Sitbon, F. (1992) Transgenic Plants Overproducing IAAxe2x80x94A Model System to Study Regulation of IAA Metabolism, Swedish University of Agricultural Sciences, Umea, Sweden).
It is an object of this invention to provide the nucleotide sequences encoding IAGlu Transferase and non-naturally occurring DNA molecules containing these sequences. An exemplary IAGlu Transferase coding sequence is that of Zea mays; as specifically exemplified herein, this sequence is presented in SEQ ID NO: 1 from nucleotide 57 to nucleotide 1472. Equivalents of the exemplified nucleotide sequence are those nucleotide sequences which encode a polypeptide with the specifically exemplified amino acid sequence given in SEQ ID NO: 2 and those nucleotide sequences which encode a polypeptide with equivalent enzymatic activity and which nucleotide sequences have substantial sequence identity (at least about 70%) to the exemplified sequence, i.e., can hybridize with the exemplified sequences under conditions of moderate or greater stringency as understood in the art.
It is a further object of this invention to provide for transcriptional expression of sequences complementary to the IAGlu Transferase coding sequences to reduce IAGlu Transferase gene expression in transgenic plants in order to down-regulate synthesis of the IAGlu Transferase in those plants, thus allowing for control of the proportions of free and bound IAA, thereby allowing for control of the growth habit of said plants. Conversely, transgenic plants which overexpress IAGlu Transferase are also taught herein. An iaglu coding sequence linked to either a regulated or a constitutive promoter can be introduced into plant tissue, and a transgenic plant regenerated, whereby control of the growth habit results from the relative overproduction of IAGlu Transferase in said plant. Overproduction of IAGlu synthetase results in loss of apical dominance, and a more prostrate plant than the wild-type parent plant.